Targeting gut microbiota–derived kynurenine to predict and protect the remodeling of the pressure-overloaded young heart

Pressure-overloaded left ventricular remodeling in young population is progressive and readily degenerate into heart failure. The aims of this study were to identify a plasma metabolite that predicts and is mechanistically linked to the disease. Untargeted metabolomics determined elevated plasma kynurenine (Kyn) in both the patient cohorts and the mice model, which was correlated with remodeling parameters. In vitro and in vivo evidence, combined with single-nucleus RNA sequencing (snRNA-seq), demonstrated that Kyn affected both cardiomyocytes and cardiac fibroblasts by activating aryl hydrocarbon receptors (AHR) to up-regulate hypertrophy- and fibrosis-related genes. Shotgun metagenomics and fecal microbiota transplantation revealed the existence of the altered gut microbiota-Kyn relationship. Supplementation of selected microbes reconstructed the gut microbiota, reduced plasma Kyn, and alleviated ventricular remodeling. Our data collectively discovered a gut microbiota–derived metabolite to activate AHR and its gene targets in remodeling young heart, a process that could be prevented by specific gut microbiota modulation.

was applied in metabolite annotation. The cutoff for annotation was set at 0.3. Statistical significance was calculated between groups using multiple t-test. Adjusted P values were generated from P values corrected by false discovery rate (FDR) based on Benjamini-Hochberg procedure. Significantly different metabolites were selected through a criteria of: (1) variable importance in projection (VIP) >1; (2)Adjusted P value <0.05.

UHPLC-MS/MS analysis
To quantify Kyn level in plasma of patients and mice, UHPLC-MS/MS analysis was

Neonatal ascending aorta constriction (nAAC) mice model
Ascending aorta constriction surgery was performed in mice pup (WT or AHR -/-) within 24 h after birth (Video 1, Data Supplement). The mice pup was firstly placed in an ice bath for 5 minutes to induce hypothermia and cardiac asystole. Then the pup was removed to an ice pack covered with gauze and was fixed with tapes. Under the microscope, a median 5mm transverse skin incision and sternum transection were made between 1 th -2 nd ribs, followed by gently pulling aside the thymus and isolating ascending aorta with two blunt forceps. Gelatin sponge could be employed to control minor hemorrhage when necessary. Inserting a 10-0 suture under the ascending aorta with the aid of a curved 32G blunt needle. Then a 3 mm 28G needle was placed on and ligated with the ascending aorta, which was not performed in sham surgery. The needle was then gently removed in nAAC surgery to induce corresponding constriction.
Closing the thorax by an 8-0 suture around the adjacent ribs, and closing the skin with three interrupted 8-0 suture. The mice pup was removed to a heat pad (37 °C) to restore its body temperature, allowing for recovery. Due to the absence of centralized pain reflexes at early postnatal age, no pain relief medications are required for P1 neonatal mice after surgery. The whole surgery could generally be performed within [10][11][12][13][14][15] minutes. All surgery in our experiments was conducted by the same surgeon with over one-year nAAC experience. Female and male mice were distributed in each group at random since gender features were not obvious in very early neonatal stage. The gender feature was determined at evaluation time points in mice after P21. The mice were harvest at indicated time points for different experiments.
RNA extraction from mice LV tissues was performed by regular Trizol method. 1 μg RNA per sample was used as input material for the RNA sample preparations.
Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample.
The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA) to select cDNA fragments of preferentially 250~300 bp in length. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. The downstream analysis was based on clean, high quality data filtered through in-house Peri scripts. Differential expression analysis was peformed using the DESeq2 R package (1.16.1). Gene with adjusted P value <0.05 and fold change >0.5 were used for following IPA analysis. Ingenuity® Pathway Analysis software was used for IPA analysis to filter the genes that putatively regulated by AHR.

Single-nucleus RNA-sequencing
Single-nucleus RNA-seq experiment of 4 human LV tissues were performed in the laboratory of NovelBio Co., Ltd (Shanghai, China). The nucleus was isolated and purified as previously described with some modifications. Briefly, the liquid nitrogen frozen LV tissues were homogenized in NLB buffer which contain 250 mM Sucrose, 10 mM Tris-HCl, 3 mM MgAc2, 0.1% Triton X-100 (SigmaAldrich, USA), 0.1 mM EDTA, 0.2U/μL RNase Inhibitor (Takara, Japan). The concentration of nucleus was adjusted to approximately 1000 nuclei/μl for snRNA-Seq. The snRNA-Seq libraries were generated using the 10X Genomics Chromium Controller Instrument and Chromium Single Cell 3' V3.1 Reagent Kits (10X Genomics, Pleasanton, USA).
Briefly, cells nuclei were loaded into each channel to generate single-cell Gel Bead-In-Emulsions (GEMs). After the RT step, GEMs were broken and barcoded-cDNA was purified and amplified. The amplified barcoded cDNA was fragmented, A-tailed, ligated with adaptors and index PCR amplified. The final libraries were quantified using the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific, USA) and the size distribution of the libraries were determined using a High Sensitivity DNA chip on a Bioanalyzer 2200 (Agilent, USA). All libraries were sequenced by Novaseq6000 (Illumina, USA) on a 150 bp paired-end run.
snRNA-seq data analysis was performed by NovelBio Bio-Pharm Technology Co.,Ltd.
with NovelBrain Cloud Analysis Platform. We applied fastp with default parameter to filter the adaptor sequence and removed the low quality reads. Then the feature-barcode matrices were obtained by aligning reads to the human genome (GRCh38_Ensembl_Ensembl100) using CellRanger v5.0.1. We applied the down sample analysis among sequenced samples according to the mapped barcoded reads per cell of each sample and finally achieved the aggregated matrix. Cells contained over 200 expressed genes and mitochondria UMI rate below 10% passed the cell quality filtering and mitochondria genes were removed in the expression Wilcoxon rank-sum test (two-tailed) was used for testing the difference between two groups and Kruskal-Wallis test was used for testing the difference between three groups.
Multiple testing was adjusted using the Benjamin-Hochberg method. Permutation multivariate analysis of variance (PERMANOVA) test was used for the difference of β diversity between the compared groups.

Probiotics administration in nAAC mice
For oral probiotics administration, each probiotics capsule containing more than were gavaged daily with 0.015ml/g probiotics slurry until euthanize, or saline as control.
To be noted, gavage in mice under 2-week age was operated with great care. Instead of using the regular steel gavage needle, 26G soft venous trocar tube was curved at tip and inserted gently beyond pharyngeal, and the gavage was controlled in a smooth and slow rate. The success of gavage was confirmed by the inflate of stomach that could be observable. Mice that died within 10 minutes after gavage procedure were attributed to gavage injury and were excluded. All mice were co-housed with their mothers and breast fed before P28, then were provided with standard fodder, in order to diminish the diet variation and its impact on gut microbiota.

Germ-free mice treatment
Germ-free (GF) mice (4-week age, 5 male and 5 female per group) were used to study the relationship of gut microbiota and blood Kyn (Supplementary Figure 11). Baseline blood samples were firstly collected through orbital vein before treatment. Then fecal microbiota transplantation (FMT) was conducted for 2 weeks in GF mice, where fecal pellets were collected from donor nAAC or sham C57BL/6 mice (4-week age) in an empty, sterile plastic cage. The gender of GF mice and nAAC/sham donor mice was also matched (5 male vs 5 female per group). The fecal supernatant processing was conducted in germ-free working hood. 50 mg collected feces were suspended in 1 ml saline. The suspension then underwent centrifuge (100×g for 2 min) to pellet large particles and collect supernatant for subsequent FMT (0.015ml/g daily). The final products were used immediately for FMT via oral gavage. After 2 weeks of FMT, a second blood collection was performed. Then the mice with nAAC FMT underwent probiotics gavage for another 2 weeks and then the third blood collection was performed. All mice were fed with sterilized water and food ad libitum.

Kyn injection in mice
L-Kynurenine (K8625, CAS:2922-83-0, Sigma) was injected intra-peritoneally every day after surgery at the dose of 50 mg/kg to induce detectable phenotypes. Same amount of saline was injected in control group.

Isolation and culture of primary neonatal mouse cardiomyocytes and cardiac fibroblasts
Primary neonatal mouse cardiomyocytes (mCM) and cardiac fibroblasts (mCF) were obtained from neonatal C57BL/6 using Neomyt kit (no.nc-6031, Cellutron). Briefly, for each time experiment, LV tissues were harvest from 10 neonatal C57BL/6 mice within 24h after birth and firstly digested for 12 min in 10 ml enzyme buffer. The supernatant was collected in a new tube. 4 ml of new enzyme buffer was added to the remaining tissues and digested for 15min. This digestion steps were repeated for 7-9 times until fully digested. The supernatant collection from each round underwent 1200 rpm, 1 min centrifuge to obtain pellets of digested cells. The pellet was resuspended in DMEM/ F12 culture medium (no.11320033, Gibco) and plated onto 6 cm culture plates.
mCF was obtained 2 hours after initial plating. The unattached cells in culture medium were transferred onto new plates to obtain mCM. Cells were cultured for 24h and then were ready for later treatment. were added into culture medium as indicated combination. Same amount of DMSO was added as control. The sequential analysis was carried out after 48h of treatment.

Isolation of cardiomyocytes and cardiac fibroblasts from 4w mice
Isolation and harvest of cardiomyocytes and cardiac fibroblasts from young mice were conducted according to a modified protocol as reported previously(52). 4-week age WT or AHR -/mice after sham or nAAC surgery, with or without Kyn injection or probiotics administration, were used for CM and CF isolation. Briefly, the mice was firstly anesthetized and fixed, the chest was then opened in a U-shaped incision. The descending aorta and inferior vena cava were transected at diaphragm level, and 5 ml EDTA buffer was quickly injected into the right ventricle with 30G needle. Then the ascending aorta was ligated with 6-0 thread above the origin of coronary artery. 5 ml EDTA buffer was injected at the apex of left ventricle, followed by 3 ml perfusion buffer and 40 ml collagenase buffer. The heart was cut off and gently pulled and pipetted into cellular debris in a 6 mm plate with 5 ml stop buffer. The cell suspension was passed through 100 μm filter and divided into two 15 ml tube. Then the suspension was allowed for gravity settling for 20 min and another three rounds of 10 min gravity settling in 4 ml perfusion buffer. CM were obtained at the bottom of the tube after final round of gravity settling. Supernatant of each round were collected together and underwent 1000 rpm, 5 min centrifuge to obtain CF pellet. The isolated CM and CF were used for further qPCR and Western blot analysis.

In vitro immunofluorescence of AHR translocation
HiPSC-CMs and hCFs were seeded in 12-well glass bottom plates (no.P24-1.5H-N, Cellvis) at the density of 10,000 cells/cm 2 . Cells were exposed to 500 μM Kyn, or The relative mRNA expression was calculated based on delta-delta CT method as previously reported (52).

Fig. S19. Schematic of FMT strategies on GF mice.
Fresh fecal pellets were collected from donor nAAC or sham C57BL/6 mice (5 male and 5 female per group) at the 4 th week after surgery in an empty, sterile plastic cage.
Then fecal supernatant was prepared in a germ-free working hood. To examine the relationship between Kyn and nAAC gut microbiota (Figure 6B), before first FMT, blood samples were firstly collected in 4-week age GF mice (pre-FMT, n=10). Then GF mice were gavaged with 0.015ml/g fecal solution daily for 2 weeks, followed by a second blood collection (post-FMT, n=10). For evaluation of the effect of probiotics ( Figure 6I), the post-FMT GF mice were then gavaged daily with 0.015ml/g probiotics slurry for 2 weeks, and the third blood collection was conducted (post-probiotics, n=10).